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Blood, Vol. 113, Issue 19, 4799-4809, May 7, 2009
Regulated release and functional modulation of junctional adhesion molecule A by disintegrin metalloproteinases
Blood Koenen et al.
113: 4799
Supplemental materials for: Koenen et al
Files in this Data Supplement:
- Figure S1. Role of ADAM10 and ADAM17 in JAM-A shedding (JPG, 82.1 KB)
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(A) Overexpression of ADAM10 and 17 in human HEK293 cells. Cells were transiently transfected with ADAM10 and ADAM17 or the empty expression vector as a control. Cell lysates (upper and middle panel) were subjected to Western blotting using ADAM17 or ADAM10 antibodies, respectively. β-actin served as a loading control. JAM-A release after overexperssion of ADAM10 and 17 is shown in Fig. 4A. (B) Downregulation of endogenous ADAM10/17 using siRNA. HUVECs were transfected with ADAM10- and ADAM17- or control-siRNA. Downregulation of ADAM10 and ADAM17 surface expression was analyzed by Western blotting. β-actin served as a loading control. Soluble JAM-A released into the conditioned media is shown in Fig. 4B (C) HEK293 cells transiently transfected with ADAM10-, ADAM17-, or control-siRNA were stimulated with PMA or left unstimulated for 2 h. Cell lysates were probed with polyclonal antibodies to human ADAM17, ADAM10, or β-actin (three upper panels). Conditioned serum-free media were subjected to Western blotting using an anti–hJAM-A monoclonal antibody (two lower panels). (D) Simultaneous downregulation of ADAM10 and ADAM17. HUVECs and HEK293 cells were treated with specific siRNA to downregulate ADAM10, ADAM17, or both proteases. Subsequently release of sJAM-A into the conditioned media was analyzed by Western blotting. (E) JAM-A shedding in adam10−∕− fibroblasts. Total cell extracts from adam10−∕− and wt MEFs were controlled for the absence and presence of ADAM10 by Western blotting using a monoclonal antibody against murine ADAM10 (upper panel). Adam10−∕− and wt MEFs were transiently transfected with JAM-A. After 48 h conditioned media and cell lysates were harvested and analyzed for the presence of sJAM-A and flJAM-A by Western blotting. Western Blot signals were quantified by densitometry and results shown as mean and SD for 3 independent experiments (lower left and right panel).
- Figure S2. Peptide mass fingerprinting of sJAM-a (JPG, 81.4 KB)
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(A) Mass spectrum of a cleavage fragment from recombinant ADAM-17–treated JAM-A.Fc that was excised from SDS-PAGE gel, trypsin-digested, and analyzed by MALDI-TOF spectrometry. Detected peptide m/z ratios are shown above the peaks. Inset: spectrum showing region from m/z ratio 499 to 904. The peak with m/z 568.10 is from an unidentified fragment which might be derived from the trypsin preparation. (B) JAM-A sequence (amino acids 28–235) in which the detected fragments and corresponding masses (in Da) are highlighted in bold black or blue.
- Figure S3. sJAM-A and neutrophil recruitment (JPG, 57.9 KB)
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(A) HUVECs and neutrophils were coincubated or incubated separately for 2 h and subsequently JAM-A surface expression on neutrophils and HUVECs was analyzed by flow cytometry. Residual surface expression on the coincubated cells was expressed as percentage of the surface expression determined for HUVECs and neutrophils that were incubated separately and statistically significant differences were indicated by asterisks. (B) HUVECs were pretreated for 1 h with an antibody to the second Ig-domain (clone 43, 10 µg/ml) of JAM-A, IgG control, or vehicle (PBS) and subsequently assayed for neutrophil transmigration in response to IL-8. Results are expressed in relation to the PBS-treated control. (C) HUVECs were left untreated or pretreated for 1 h with recombinant catalytic domain of ADAM17 (10 ng/ml). The protease and its shedding products were then removed by washing or left with the cells that were subsequently assayed for IL-8–induced neutrophil transmigration. Results are expressed in relation to the untreated control. (D) Murine leukocytes form peripheral blood were incubated with human IgG control or JAM-A.Fc in the absence or presence of an equimolar amount of monovalent sJAM and subsequently bound Fc-molecules on neutrophils were detected by flow cytometry using an anti-human Fc PE-conjugate. (E) JAM-A.Fc or human IgG (60 µg) were intravenously administered to mice (n = 5 per group), and 1 h later IL-8 or vehicle control was injected into experimentally induced air pouches. 4 h after IL-8 injection, neutrophil recruitment into the air pouches was determined by flow cytometric analysis of the differentially labeled leukocyte populations. The asterisks in B and D indicate statistically significant changes in neutrophil infiltration compared to the IgG-treated control (p<0.05).
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